High Selectivity of an α-Conotoxin LvIA Analogue for α3β2 Nicotinic Acetylcholine Receptors Is Mediated by β2 Functionally Important Residues

Abstract

The α3β2 and α3β4 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and peripheral nervous systems, playing critical roles in various physiological processes and in such pathologies as addiction to nicotine and other drugs of abuse. α-Conotoxin LvIA, which we previously isolated from Conus lividus, modestly discriminates α3β2 and α3β4 rat nAChRs exhibiting a ∼17fold tighter binding to the former. Here, alanine scanning resulted in two more selective analogues [N9A]LvIA and [D11A]LvIA, the former having a >2000-fold higher selectivity for α3β2. The determined crystal structures of [N9A]LvIA and [D11A]LvIA bound to the acetylcholinebinding protein (AChBP) were followed by homologous modeling of the complexes with the α3β2 and α3β4 nAChRs and by receptor mutagenesis, which revealed Phe106, Ser108, Ser113, and Ser168 residues in the β2 subunit as essential for LvIA binding. These results may be useful for the design of novel compounds of therapeutic potential targeting α3β2 nAChRs.

Characterization of an α 4/7-Conotoxin LvIF from Conus lividus That Selectively Blocks α3β2 Nicotinic Acetylcholine Receptor

“…[K11A]TxIB displayed an obvious block on rα7 nAChR, while the wild-type TxIB has no inhibition on rα7 nAChR [42]. Similar situation occurred for α-CTx TxID vs. [S9K]TxID and α-CTx LvIA vs. [N9A]LvIA [11,35]. Therefore, a key single amino acid substitution could affect the activity and selectivity of an α-CTx, which occurred similarly for GIC and LvIF in this work.…”

Potency- and Selectivity-Enhancing Mutations of Conotoxins for Nicotinic Acetylcholine Receptors Can Be Predicted Using Accurate Free-Energy Calculations

“…In this study, we use FEP to retrospectively predict potency and selectivity data for an archetypical system, the α-CTX LvIA from Conus lividus , which is naturally 18-fold more selective for the α3β2 nAChR than the highly similar α3β4 nAChR [ 26 ] ( Figure 2 C,D). The α3β2 nAChR is involved in a variety of physiological processes and the α3β4 nAChR is implicated in nicotine addiction [ 11 ].…”

“…The α3β2 nAChR is involved in a variety of physiological processes and the α3β4 nAChR is implicated in nicotine addiction [ 11 ]. This conotoxin and nAChR pair serves as a rigorous test for FEP selectivity calculations because the ECDs of the α3β2 and α3β4 nAChRs are 68% identical and point mutants of LvIA with a wide range of selectivity levels, some of which are counterintuitive, have been identified [ 26 ]. For example, LvIA[N9A] is >2000-fold more selective for the α3β2 nAChR than the α3β4 nAChR, although this mutant does not make significantly different contacts between the subtypes [ 26 ] ( Figure 2 C,D).…”

“…This conotoxin and nAChR pair serves as a rigorous test for FEP selectivity calculations because the ECDs of the α3β2 and α3β4 nAChRs are 68% identical and point mutants of LvIA with a wide range of selectivity levels, some of which are counterintuitive, have been identified [ 26 ]. For example, LvIA[N9A] is >2000-fold more selective for the α3β2 nAChR than the α3β4 nAChR, although this mutant does not make significantly different contacts between the subtypes [ 26 ] ( Figure 2 C,D). We begin by examining the suitability of AChBP/α-CTX complexes as templates for nAChR/α-CTX homology models by using FEP and MM-GB/SA to retrospectively predict radioligand binding data for the conotoxins LvIA and GIC at Ac-AChBP and the conotoxin LsIA at Ls-AChBP [ 13 , 26 , 27 ] ( Figure 2 E).…”

“…For example, LvIA[N9A] is >2000-fold more selective for the α3β2 nAChR than the α3β4 nAChR, although this mutant does not make significantly different contacts between the subtypes [ 26 ] ( Figure 2 C,D). We begin by examining the suitability of AChBP/α-CTX complexes as templates for nAChR/α-CTX homology models by using FEP and MM-GB/SA to retrospectively predict radioligand binding data for the conotoxins LvIA and GIC at Ac-AChBP and the conotoxin LsIA at Ls-AChBP [ 13 , 26 , 27 ] ( Figure 2 E). Second, we build homology models of the α3β2 and α3β4 nAChRs and retrospectively test the accuracy of FEP and MM-GB/SA in predicting the potency and selectivity of a set of point mutations of LvIA at these subtypes [ 26 ].…”